The proposed project is designed to continue study of the comparative structure and binding site topography of various high-affinity steroid-binding proteins. We will utilize: a) affinity-labeling steroids (site-directed irreversible inhibitors) with subsequent identification of alkylated residues and sequencing of the peptides that contain them, b) determination of primary structure, and c) x-ray crystallography. In addition, where these proteins are enzymes, we will: a) determine stereospecificity of hydrogen transfer to cofactor, b) evaluate spatial relationships of steroid and cofactor as they undergo the reversible binding step at the active site, and c) identify those residues that proximate the site of the catalytic event and presumably play a role in inducing the transition state. Steroid dehydrogenases to be used as models include human placental estradiol 17Beta-dehydrogenase, the horse placental epimeric dehydrogenases (estradiol 17Alpha- and 17Beta- dehydrogenase), rat ovarian 20Alpha-dehydrogenase and a 20-dehydrogenase from pig or cow gonad. In addition, affinity-labeling studies of chicken oviduct progesterone receptor and possibly, calf uterine estradiol receptor will be done. Currently, no real evidence exists as to how the evolutionary process has structured a steroid binding site. Structural studies, similar to those proposed in this project have been carried out with the 2-hydroxyacid dehydrogenases and have provided "in depth" understanding of binding site structure and merchanisms pertinent to the catalytic event. They have been worth the effort. We submit that the same is true in terms of those macromolecules that bind and catalyze reactions involving steroids.